Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins

  title={Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins},
  author={Gero Miesenböck and Dino A. De Angelis and James E. Rothman},
In neural systems, information is often carried by ensembles of cells rather than by individual units. Optical indicators provide a powerful means to reveal such distributed activity, particularly when protein-based and encodable in DNA: encodable probes can be introduced into cells, tissues, or transgenic organisms by genetic manipulation, selectively expressed in anatomically or functionally defined groups of cells, and, ideally, recorded in situ, without a requirement for exogenous cofactors… 

pHTomato: A genetically-encoded indicator that enables multiplex interrogation of synaptic activity

A bright, red, pH-sensitive fluorescent protein, pHTomato, that can be used in parallel with green probes to monitor neuronal activity and a close size matching between pre- and postsynaptic compartments, as well as interesting target cell–dependent regulation of presynaptic vesicle pools are observed.

Visualizing Presynaptic Calcium Dynamics and Vesicle Fusion with a Single Genetically Encoded Reporter at Individual Synapses

A single molecule reporter capable of concurrent imaging of both presynaptic calcium influx and exocytosis is constructed by fusion of sypHy, the vesicle associated protein synaptophysin containing a GFP-based pHluorin sensor, with the red-shifted GECI R-GECO1.

Studying vesicle cycling in presynaptic terminals using the genetically encoded probe synaptopHluorin

This work describes how a group of GFP-derived probes called pHluorins can be used in rodent hippocampal neurons to monitor the synaptic vesicle cycle in real time and to obtain mechanistic insights about it.

Survey of Red Fluorescence Proteins as Markers for Secretory Granule Exocytosis

Tandem dimer (td)-mOrange2 was identified as well-targeted, bright, slowly bleaching and pH-sensitive FP that performed similar to EGFP, and their performance in detecting exocytosis in live cells is reported.

Imaging Synaptic Vesicle Exocytosis-Endocytosis with pH-Sensitive Fluorescent Proteins.

The process of imaging synaptic vesicle endocytosis in response to extracellular stimulation in dissociated neuronal cultures of hippocampal neurons obtained from rats-also applicable to mice- is described using pHluorin-tagged vesicular glutamate transporter-1 as a reporter.

Novel pH-Sensitive Lipid Based Exo-Endocytosis Tracers Reveal Fast Intermixing of Synaptic Vesicle Pools

The development of a new class of fluorescent probes based on pH-sensitive organic dyes covalently bound to lipids, providing a promising complementary assay to genetically encoded fluorescent probes is described, providing strong evidence for a highly dynamic and use-dependent control of the fractions of releasable or resting SVs.

Study of synaptic vesicle cycling during exo- and endocytosis using optical approaches

It is concluded that functional block of previously used release sites rather than insufficient vesicle supply accounts for the fast release depression after blocking endocytosis, implying an important role of dynamin for sustained synaptic transmission at high rates beyond its well-known role in mediating fission, a late step of endocyTosis.

Intracellular pH measurements in vivo using green fluorescent protein variants.

  • K. Nehrke
  • Biology
    Methods in molecular biology
  • 2006
This chapter presents a technique whereby the optically transparent nematode Caenorhabditis elegans, engineered to express a fluorescent pH indicator protein, can be used to study how intracellular pH fluctuates in response to environmental and/or experimental challenge.



Patterns of synaptic activity in neural networks recorded by light emission from synaptolucins.

Fusion proteins of Cypridina luciferase and synaptotagmin-I or VAMP-2/synaptobrevin were expressed in cultured hippocampal neurons with the help of viral vectors and formed light-emitting complexes with their cognate luciferin, which was added to the extracellular medium.

Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin

New fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations are constructed and dubbed ‘cameleons’.

Fluorescent probes of cell signaling.

  • R. Tsien
  • Chemistry, Biology
    Annual review of neuroscience
  • 1989
The earliest developed and most straight­ forward uses of fluorescent groups are simply as positional tags or markers, while a second type of application of fluorescent probes involves attachment of the fluorophore to a purified macromolecule to sense conformational change of the latter.

Capacitance measurements reveal stepwise fusion events in degranulating mast cells

The results show that degranulation occurs spontaneously and reproducibly if the GTP analogue, GTP-γ-S, and Mg-ATP are present in the pipette filling solution and that guanine nucleotide regulatory proteins are involved in the control of this process.

Detection in Living Cells of Ca2+-dependent Changes in the Fluorescence Emission of an Indicator Composed of Two Green Fluorescent Protein Variants Linked by a Calmodulin-binding Sequence

This observation suggests that the activity of a calmodulin target with a typical 1 nmaffinity for (Ca2+)4-calmodulin is responsive to changes in the intracellular Ca2+ concentration over the physiological range.


Abstract— In the jellyfish Aequorea, the green‐fluorescent protein (GFP) functions as the in vivo bio‐luminescence emitter via energy transfer from the photoprotein aequorin. Accumulated evidence has

A view of acidic intracellular compartments

To better understand the function of acidic compartments, investigators have studied H § gradients in the living cell using vital pH indicators and recent advances in immunocytochemistry, flow cytometry, and videoenhanced microscopy offer the interested investigator new opportunities for using these indicators to "capture the H § gradient as it exists in theliving cell and detect the chemical reactions that occur in that environment.

The molecular structure of green fluorescent protein

The crystal structure of recombinant wild-type green fluorescent protein (GFP) has been solved to a resolution of 1.9 Å by multiwavelength anomalous dispersion phasing methods and the identification of the dimer contacts may allow mutagenic control of the state of assembly of the protein.